The current project seeks to provide new information concerning the molecular mechanism of Ca++ as a regulator of pancreatic exocrine secretion. A well-established regulatory pathway is the protein kinase-catalyzed phosphorylation of cellular substrate proteins, with subsequent alterations in the functional properties of those proteins. Recent work has indicated that Ca++ may operate through this type of mechanism in certain tissues. Our preliminary studies indicate the presence of a phospholipid-sensitive Ca++ dependent protein kinase (PL/Ca++ PK) in pancreas. We have also recently described endogenous substrate proteins for this protein kinase in pancreas, using SDS-polyacrylamide gel electrophoresis/radioautography. Ca++ is accepted as a mediator of pancreatic exocrine secretion stimulated by some agents (cholinergic agonists, cholecystokinin), and acinar cell stimulation by these agents also results in membrane phospholipid turnover, which appears to be involved in activation of PL/Ca-PK. It thus seems that this phospholipid-sensitive kinase could mediate some of the effects of Ca++ as a modulator of pancreatic secretion. This project deals with the purification and detailed study of pancreatic PL/Ca-PK and its substrate proteins, including localization of these proteins to specific subcellular populations including zymogen granule membrane, microsomal, plasma membrane and cytosolic fractions. Additional studies will involve intact pancreatic acinar cells. After 32P-labeling of ATP pools, cells will be stimulated with various secretogogues, both those utilizing Ca++ as a possible messenger and those which appear to involve mediators such as cyclic AMP. Cells will then be examined for endogenous protein phosphorylation subsequent to the secretory stimulus, and attempts made to correlate secretion with protein phosphorylation.. The project proposed will, therefore, concentrate on a new mechanism for Ca++ as a secretory regulator. Hopefully, knowledge obtained with these studies will contribute to the understanding of normal pathways of control in the pancreatic acinar cell, and provide some basis for suggesting points of pathologic lesion in digestive diseases, as well as conditions such as cystic fibrosis.